The invention discloses an in vitro reconstruction human red blood cell substitute and a preparation method thereof. A low-seepage method is used for performing in vitro reconstruction; in a low-seepage environment (containing rich hemoglobin), temporary holes occur in cytomembrane carriers; through the inside and outside hemoglobin concentration differences in the cytomembrane carriers, the hemoglobin enters the carriers through the temporary holes; then, the temporary holes in the cytomembrane can be sealed again through the flowability of the cytomembrane; the hemoglobin entering the cellscan be coated; then, the relative loading rate of the hemoglobin of the reconstruction human red blood cells is detected by an ELIASA; the form and the size of the reconstruction human red blood cellsare observed by a phase contrast microscope; the oxygen carrying function of the reconstruction human red blood cells is detected by a blood oxygen analysis instrument. A series of reaction after theblood infusion due to immunogenicity can be reduced; the in vitro reconstruction human red blood cell substitute can be applied to clinic first aid and the like. A proper amount of 2,3-DPG, ATP and the like are added into the hemoglobin liquid for loading for regulating the oxygen carrying function of the reconstruction human red blood cells, so that the blood volume of a hypoxic-ischemic patientis improved; meanwhile, the oxygen lack state in the body of the patient is relieved.